Linking Protein Dynamics to Hydrogen Tunneling

将蛋白质动力学与氢隧道联系起来

• 批准号: 0446395
• 负责人: Judith Klinman
• 金额: $ 96.03万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-15 至 2011-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0446395&HistoricalAwards=false
• 关键词: Linking; Protein; Dynamics; Hydrogen; Tunneling

The relationship between protein motions and catalytic efficiency is one of the least understood aspects of the enormous catalytic power of enzymes. Our ability to describe and predict this relationship has wide ranging implications for the de novo design of new protein catalysts. This project is focused on a highly related family of enzymes that catalyze alcohol oxidation (alcohol dehydrogenases, ADHs) that function in the range of 5C (psychrophilic ADH), 25C (mesophilic ADH) and 70C (thermophilic ADH). In each case, the ADH catalyzes the transfer of one hydrogen plus two electrons (hydride ion) from substrate to cofactor. Previous work has shown first, that these reactions occur with substantial tunneling of the hydride ion and second, that hydrogen tunneling must be linked to dynamical features within the protein. The comparison of proteins that function in different temperature niches, with different inherent dynamical and stability properties, provides a powerful experimental handle in the efforts to link protein motions and catalysis. The project will combine a range of kinetic and spectroscopic experiments that allow the P.I. to examine the properties of hydrogen transfer and protein dynamical features, respectively. Kinetic probes of hydrogen transfer will include the temperature dependence of rate and kinetic isotope effects as well as the relationship of this behavior among the three isotopes of hydrogen (protium, deuterium and tritium). Spectroscopic probes of protein dynamics will include the use of hydrogen/deuterium exchange, linked to mass spectrometry, protein fluorescence and nuclear magnetic resonance.Broader Impacts: This work is at the cutting edge of enzymology and will provide information that is directly relevant to the de novo design of new catalysts. The project is training a cadre of young personnel at different stages of careers. The project will involve training of the scientific workforce. There will be opportunities for undergraduate and graduate student research. In addition, mentoring of postdoctoral associates and beginning faculty members is an integral part of this research.

蛋白质运动和催化效率之间的关系是酶巨大催化能力中最不为人所知的方面之一。我们描述和预测这种关系的能力对于新型蛋白质催化剂的从头设计具有广泛的影响。该项目专注于催化酒精氧化的高度相关的酶家族（酒精脱氢酶，ADH），其在 5C（嗜冷 ADH）、25C（嗜温 ADH）和 70C（嗜热 ADH）范围内发挥作用。在每种情况下，ADH 都会催化一个氢加两个电子（氢负离子）从底物到辅因子的转移。先前的工作首先表明，这些反应是在氢负离子大量隧道效应的情况下发生的，其次，氢隧道效应必须与蛋白质内的动力学特征相关。对在不同温度环境中发挥作用、具有不同的固有动力学和稳定性特性的蛋白质进行比较，为将蛋白质运动和催化联系起来提供了强大的实验手段。该项目将结合一系列动力学和光谱实验，使 P.I.分别检查氢转移的性质和蛋白质动力学特征。氢转移的动力学探针将包括速率和动力学同位素效应的温度依赖性，以及氢的三种同位素（氕、氘和氚）之间这种行为的关系。蛋白质动力学的光谱探针将包括使用氢/氘交换，与质谱、蛋白质荧光和核磁共振相关。更广泛的影响：这项工作处于酶学的前沿，并将提供与酶学研究直接相关的信息。新型催化剂的创新设计。该项目正在培养处于不同职业阶段的青年骨干人才。该项目将涉及对科学人员的培训。本科生和研究生将有机会进行研究。此外，对博士后和新教师的指导也是这项研究的一个组成部分。